Conotoxin (CTx, Conopeptide) is secreted by Conus, a kind of carnivore mollusc living in tropical seas, which has special function of regulating various ion channels, and shows important value in clinic. Conotoxin usually contains 10-46 amino acids enriching with disulfide bonds, has strong biological activity, can specifically act to receptors and ion channels on animal cell membrane, specially has relatively high selectivity to voltage-gated or ligand-gated ion channels (including few G-protein associated receptors, etc.). Conotoxin can be classified into different gene families according to similarity of precursor protein endoplasmic reticulum targeting sequence and cysteine pattern. So far, all known conotoxins can be classified into 19 superfamilies, i.e., A, B, C, D, S, M, I1, I2, I3, J, L, O1, O2, O3, P, T, V, Y, K (Sulan Luo, Sean Christensen, Dongting Zhangsun, Yong Wu, Yuanyan Hu, Xiaopeng Zhu, Sandeep Chhabra, Raymond S. Norton, and J. Michael McIntosh. A Novel Inhibitor of α9α10 Nicotinic Acetylcholine Receptors from Conus vexillum Delineates a New Conotoxin Superfamily. PLoS ONE, (2013) 8(1): e54648 (1-10); Kaas Q, Yu R, Jin A H, Dutertre S and Craik D J. ConoServer: updated content, knowledge, and discovery tools in the conopeptide database. Nucleic Acids Research (2012) [Ahead of print]; Ye M, Khoo K K, Xu S, Zhou M, Boonyalai N, Perugini M A, Shao X, Chi C, Galea C A, Wang C & Norton R S. A helical conotoxin from Conus imperialis has a novel cysteine framework and defines a new superfamily. Journal of Biological Chemistry (2012) 287, 14973-14983). Conotoxin can be classified into pharmacological families α, ω, μ, δ and so on according to receptor target thereof. According to receptor target type, each superfamily of conotoxin can further be classified into α, αA, κA (A-superfamily), ω, δ, κ, μO (O-superfamily), μ, ψ, κM (M-superfamily), etc. (subtypes).
Wherein, α-conotoxin is a nicotine acetylcholine receptor (nAChRs) subtype specific blocking agent that is the one with the best selectivity known in the art. Hence, α-conotoxin and its action target nAChRs are of very important value in studying mechanisms of many diseases as well as research and development of drugs. α-Conotoxin is one of the earliest known conotoxins, has a relatively small molecular weight, usually consists of 12-19 amino acid residues, and is rich in disulfide bonds. There are many kinds of α-conotoxins with diverse activities and complicated structure changes. The α-conotoxins can be classified according to their highly conservative signal peptide sequences, pharmacological activities and cysteine patterns. The cysteine pattern of α-conotoxin is CC-C-C, in which the linkage mode of disulfides of natural peptides is C1-C3 and C2-C4, which is called as globular isomer, and 2 loops are formed between disulfide bonds. The α-conotoxin linear peptides containing 4 cysteines usually generate 3 isomers after oxidation and folding, besides natural peptide disulfide bond linkage mode of C1-C3 and C2-C4 (globular isomer), other two isomers are separately ribbon isomer and bead isomer. The ribbon isomer has linkage mode of disulfides as C1-04 and C2-C3; while the bead isomer has linkage mode of disulfides as C1-02 and C3-C4. The globular isomer has complete biological activity, the ribbon isomer exhibits biological activity sometimes via different action mechanism, while the bead isomer usually has a reduced activity. There are 2 loops formed between disulfide bonds, α-conotoxins can be classified into many subfamilies such as α3/5, α4/7, α4/6, α4/4 and α4/3 according to number of amino acids between the 2nd and 3rd cysteines and between the 3rd and the 4th cysteines, and the differences in features and residue composition of each loop form the basis that different conotoxins act on different receptor subtypes (Ulens C, Hogg R C, Celie P H, et al. Structural determinants of selective alpha-conotoxin binding to a nicotinic acetylcholine receptor homolog AChBP[J]. Proc Natl Acad Sci USA 2006; 103: 3615-20; McIntosh, J. M.; Santos, A. D.; Olivera, B. M., Conus peptides targeted to specific nicotinic acetylcholine receptor subtypes. Annual review of biochemistry 1999, 68, 59-88; Terlau, H.; Olivera, B. M., Conus venoms: a rich source of novel ion channel-targeted peptides. Physiological reviews 2004, 84 (1), 41-68. Gehrmann J, Alewood P F, Craik D J. Structure determination of the three disulfide bond isomers of alpha-conotoxin GI: a model for the role of disulfide bonds in structural stability. J Mol Biol. 1998, 278(2):401-15; Grishin A A, Wang C I, Muttenthaler M, Alewood P F, Lewis R J, Adams D J. Alpha-conotoxin AuIB isomers exhibit distinct inhibitory mechanisms and differential sensitivity to stoichiometry of alpha3beta4 nicotinic acetylcholine receptors. J Biol Chem. 2010, 285 (29): 22254-63).
Nicotine acetylcholine receptors (nAChRs) are membrane proteins that are prevalent in animal kingdom and have important physiological actions and clinical research significance, and they are the earliest receptors found by human and can be classified into two groups: muscular type acetylcholine receptors and neurologic type acetylcholine receptors. The nAChRs are allosteric membrane proteins on cell membrane, mediate many physiological functions of central and peripheral nervous systems, including learning, memory, addiction, response, and analgesia and motion control. The nAChRs activate release of many neurotransmitters such as dopamine, noradrenaline, serotonin, γ-aminobutyric acid. It is confirmed that nAChRs are critical targets for screening medicines in diagnosis and treatment of a large group of important diseases, and these diseases include pains, addiction of tobacco, alcohol and drugs, amentia, dementia, schizophrenia, disorder of central nerves, epilepsy, Parkinson's disease, mental diseases, neuromuscular blockade, myasthenia gravis, depression, hypertension, arrhythmia, asthma, muscular flaccidity, apoplexy, breast cancer and lung cancer. So far, there is no medicine for symptomatic treatment of these diseases. Common non-selective nAChRs agonists such as nicotine could relieve symptoms of the above nerve diseases, but they have strong side-effects on heart and gastrointestinal tract and addiction. Hence, the key for treatment of the above diseases is to develop ligand medicines having high selectivity on various subtypes of nAChRs. (Livett B G, Sandal) DW, Keays D, Down J, Gayler K R, Satkunanathan N, Khalil Z. Therapeutic applications of conotoxins that target the neuronal nicotinic acetylcholine receptor. Toxicon, 2006, 48(7):810-829; Taly A, Corringer P J, Guedin D, Lestage P, Changeux J P. Nicotinic receptors: allosteric transitions and therapeutic targets in the nervous system. Nat Rev Drug Discov. 2009, 8(9): 733-50; Layla A, McIntosh J M. Alpha-conotoxins as pharmacological probes of nicotinic acetylcholine receptors [J]. Acta Pharmacol Sin 2009 June; 30 (6): 771-783.).
However, the precondition for developing such medicines is to obtain selective compounds capable of specifically binding to various subtypes of nAChRs, which can be used as tool drugs to study and identify fine composition and physiological functions of various subtypes, or can be directly used as therapeutic drugs for treatment of associated diseases. In addition, the activation of nicotine acetylcholine receptor on tumor cell membrane of breast cancer and small cell lung cancer can promote tumor cell proliferation, so blocking the activation of these receptors with drugs can effectively be used to perform early diagnosis or treatment of these catastrophic cancers.
The nAChRs are assembled with different α and β subunits to form many subtypes, and each subtype has distinctive pharmacological features, among which muscular acetylcholine receptors consist of 5 subunits, including two α1 subunits, one β subunit, one δ subunit and one γ or ε subunit, and whether it is γ or ε subunit depends on whether it is fetal or adult acetylcholine receptor. The subtypes of neurologic nAChRs of mammals are far more complex than muscular nAChRs, have at least 8 α subunits, 3 β subunits, which separately are: α2-α7, α9, α10 (α8 in chicken), and β2-β4. Wherein, α2, α3 and α4 can separately bind to β2 or β4 to form functional receptors, such as α2β2, α3β2, α2β4; α9 and α10 are bound to form functional receptor α9α10 nAChRs. In addition, α7 and α9 can form homologous multimers. Due to lack of ligand compounds with high selectivity to various subtypes, there are a lot of challenges to study and illustrate the fine structure and function of various nAChRs subtypes.
Drug addiction is both medical challenge and social problem. Smoking addiction is caused with nicotine in tobacco, of which receptors in vivo are nicotine acetylcholine receptors (nAChRs) (Azam L, McIntosh J M. Alpha-conotoxins as pharmacological probes of nicotinic acetylcholine receptors. Acta Pharmacol Sin. 2009; 30(6): 771-783). Some researches show that the expression of nAChRs of dopaminergic (DA) neuron are drug action targets for treatment of neuropsychological diseases, such as addiction of nicotine, morphine, cocaine, Parkinson's disease, dementia, schizophrenia, depression (Larsson, A.; Jerlhag, E.; Svensson, L.; Soderpalm, B.; Engel, J. A., Is an alpha-conotoxin MII-sensitive mechanism involved in the neurochemical, stimulatory, and rewarding effects of ethanol? Alcohol 2004, 34 (2-3), 239-50. Jerlhag, E.; Egecioglu, E.; Dickson, S. L.; Svensson, L.; Engel, J. A., Alpha-conotoxin MII-sensitive nicotinic acetylcholine receptors are involved in mediating the ghrelin-induced locomotor stimulation and dopamine overflow in nucleus accumbens. European neuropsychopharmacology, 2008, 18 (7), 508-18). The α-conotoxin-MII capable of blocking α3β2 and α6β2* nAChRs can partially and differentially block dopamine release from striatal synaptosomes, and presynaptic nAChRs contains at least 2 subtypes, i.e., MII-sensitive type and MII-nonsensitive type, that are capable of regulating DA release of dopamine neurons. (Kaiser S A, Soliakov L, Harvey S C, Luetje C W, Wonnacott S. Differential inhibition by α-conotoxin-MII of the nicotinic stimulation of [3H]-dopamine release from rat striatal synaptosomes and slices. J Neurochem 1998; 70: 1069-76). Some new reports show that blocking nAChRs containing α3β4 or α6β2 can effectively prevent onset of smoking addiction and morphine addiction, significantly inhibiting desire for smoking and drug (Brunzell D H, Boschen K E, Hendrick E S, Beardsley P M, McIntosh J M. Alpha-conotoxin MII-sensitive nicotinic acetylcholine receptors in the nucleus accumbens shell regulate progressive ratio responding maintained by nicotine. Neuropsychopharmacology, 2010; 35(3):665-673.).
In addition, DA neuron has a very high expression amount of nAChRs containing α6 subunit, and due to the lack of pharmacologic molecular probe specific to α6* nAChRs, the important action mechanism of α6 nAChR in addiction is still not clear. The α6β2*-nAChRs subtype on striate body of mammal brain is considered as drug action target for treatment of smoking addiction and drug addiction (Exley, R.; Clements, M. A.; Hartung, H.; McIntosh, J. M.; Cragg, S. J., Alpha6-containing nicotinic acetylcholine receptors dominate the nicotine control of dopamine neurotransmission in nucleus accumbens. Neuropsychopharmacology 2008, 33 (9), 2158-66). The α6 subtype is not broadly distributed in brain, but enriched in midbrain dopaminergic neuron region, while this region is closely related to happiness, reward and mood control, and this means α6* nAChRs play a vital role in drug addiction and mood control (Yang, K. C., G. Z. Jin, et al. (2009). Mysterious alpha6-containing nAChRs: function, pharmacology, and pathophysiology. Acta Pharmacol Sin 30(6): 740-751. Klink, R.; de Kerchove d'Exaerde, A.; Zoli, M.; Changeux, J. P., Molecular and physiological diversity of nicotinic acetylcholine receptors in the midbrain dopaminergic nuclei. The Journal of neuroscience, 2001, 21 (5), 1452-63. Azam, L.; Winzer-Serhan, U. H.; Chen, Y.; Leslie, F. M., Expression of neuronal nicotinic acetylcholine receptor subunit mRNAs within midbrain dopamine neurons. The Journal of comparative neurology 2002, 444 (3), 260-74. Champtiaux, N.; Gotti, C.; Cordero-Erausquin, M.; David, D. J.; Przybylski, C.; Lena, C.; Clementi, F.; Moretti, M.; Rossi, F. M.; Le Novere, N.; McIntosh, J. M.; Gardier, A. M.; Changeux, J. P., Subunit composition of functional nicotinic receptors in dopaminergic neurons investigated with knock-out mice. The Journal of neuroscience, 2003, 23 (21), 7820-9. Pons, S.; Fattore, L.; Cossu, G.; Tofu, S.; Porcu, E.; McIntosh, J. M.; Changeux, J. P.; Maskos, U.; Fratta, W., Crucial role of alpha4 and alpha6 nicotinic acetylcholine receptor subunits from ventral tegmental area in systemic nicotine self-administration. The Journal of neuroscience, 2008, 28 (47), 12318-27). The α6* nAChRs are also expressed in catecholaminergic nuclei and retina (Le Novere, N.; Zoli, M.; Changeux, J. P., Neuronal nicotinic receptor alpha 6 subunit mRNA is selectively concentrated in catecholaminergic nuclei of the rat brain. The European journal of neuroscience 1996, 8 (11), 2428-39. Vailati, S.; Hanke, W.; Bejan, A.; Barabino, B.; Longhi, R.; Balestra, B.; Moretti, M.; Clementi, F.; Gotti, C., Functional alpha6-containing nicotinic receptors are present in chick retina. Molecular pharmacology 1999, 56 (1), 11-9.). The α6β2* nAChRs show function of regulating dopamine release, and the amount of α6β2* nAChRs significantly decreases in primate animal model of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and human Parkinson's disease model (Champtiaux, N.; Han, Z. Y.; Bessis, A.; Rossi, F. M.; Zoli, M.; Marubio, L.; McIntosh, J. M.; Changeux, J. P., Distribution and pharmacology of alpha 6-containing nicotinic acetylcholine receptors analyzed with mutant mice. The Journal of neuroscience, 2002, 22 (4), 1208-17. Quik, M.; Polonskaya, Y.; Kulak, J. M.; McIntosh, J. M., Vulnerability of 125I-alpha-conotoxin MII binding sites to nigrostriatal damage in monkey. The Journal of neuroscience, 2001, 21 (15), 5494-500. Quik, M.; Bordia, T.; Forno, L.; McIntosh, J. M., Loss of alpha-conotoxin MII- and A85380-sensitive nicotinic receptors in Parkinson's disease striatum. Journal of neurochemistry 2004, 88 (3), 668-79). Hence, α6/α3β2β3 nAChRs-specific blocking agents are valuable tools for studying and explaining physiological functions of α6* nAChRs in different tissues, medicaments for treatment of associated diseases such as addiction, Parkinson's diseases, or tool drugs for screening such medicaments.
New research shows that blocking nAChRs containing α3β4 can effectively prevent onset of smoking addiction, morphine and cocaine addiction, significantly inhibit desire for smoking and drug (Brunzell D H, Boschen K E, Hendrick E S, Beardsley P M, McIntosh J M. Alpha-conotoxin MII-sensitive nicotinic acetylcholine receptors in the nucleus accumbens shell regulate progressive ratio responding maintained by nicotine, Neuropsychopharmacology, 2010, 35(3):665-73).
Surveys show that about ⅙ population suffer pains, including arthritis, neuralgia, sore pain, in which neuralgia affects 4-8% of population, and neuralgia may be caused by alcoholism, ischioneuralgia, cancers and cancer chemotherapy, diabetes mellitus, prosopalgia, sclerosis, herpes zoster, mechanical injury. The nAChRs containing α3-subunit, including α3β2 and α3β4 subtypes, are mainly expressed in peripheral nervous system, and also distributed in central nervous system, and are targets for action of neuralgic medicines. The α-conotoxin capable of blocking α3β2 or α3β4 nAChRs shows excellent analgesic activity to many chronic pains in clinic, and is not addicted. Chronic pains are a health challenge worldwide, and in urgent need of new therapeutic drugs (Napier, I. A.; Klimis, H.; Rycroft, B. K.; Jin, A. H.; Alewood, P. F.; Motin, L.; Adams, D. J.; Christie, M. J., Intrathecal α-conotoxins Vc1.1, AuIB and MII acting on distinct nicotinic receptor subtypes reverse signs of neuropathic pain. Neuropharmacology 2012, 62 (7), 2202-2207. Blyth, F. M.; March, L. M.; Brnabic, A. J.; Jorm, L. R.; Williamson, M.; Cousins, M. J., Chronic pain in Australia: a prevalence study. PAIN 2001, 89 (2-3), 127-34. Cousins, M. J.; Brennan, F.; Carr, D. B., Pain relief: a universal human right. PAIN 2004, 112 (1-2), 1-4. Eisenberg, E.; McNicol, E. D.; Carr, D. B., Efficacy and safety of opioid agonists in the treatment of neuropathic pain of nonmalignant origin: systematic review and meta-analysis of randomized controlled trials. JAMA: the journal of the American Medical Association 2005, 293 (24), 3043-52.).
The α3β4 nAChRs are main acetylcholine receptor subtypes in sensory and autonomic nerve centers. The α3β4 nAChRs are also branches of central nervous system (CNS) neurons, such as habenula extended to central and back marrow, and relate to addiction of nicotine and other abuse drugs (Millar, N. S.; Gotti, C., Diversity of vertebrate nicotinic acetylcholine receptors. Neuropharmacology 2009, 56 (1), 237-46; Tapper, A. R.; McKinney, S. L.; Nashmi, R.; Schwarz, J.; Deshpande, P.; Labarca, C.; Whiteaker, P.; Marks, M. J.; Collins, A. C.; Lester, H. A., Nicotine activation of alpha4* receptors: sufficient for reward, tolerance, and sensitization. Science 2004, 306 (5698), 1029-32.). The α3β4 nAChR relates to limbic dopamine pathways, and plays very important role to reward effects generated by abuse substances (such as drugs). In β4-subunit knockout mice, the motion and reward effects caused by nicotine decrease significantly, which suggests the important effects of α3β4 nAChR on nicotine addiction in CNS (Sales, R., Sturm, R., Boulter, J., and De Biasi, M. (2009) Nicotinic receptors in the habenulo-interpeduncular system are necessary for nicotine withdrawal in mice. J. Neurosci. 29, 3014-3018). The α3β4 nAChRs also play a very important role in threatening response, and significantly affect the regulation of glutamic acid and the release of noradrenaline (Zhu, P. J.; Stewart, R. R.; McIntosh, J. M.; Weight, F. F., Activation of nicotinic acetylcholine receptors increases the frequency of spontaneous GABAergic IPSCs in rat basolateral amygdala neurons. Journal of neurophysiology 2005, 94 (5), 3081-91. Alkondon, M.; Albuquerque, E. X., A non-alpha7 nicotinic acetylcholine receptor modulates excitatory input to hippocampal CA1 interneurons. Journal of neurophysiology 2002, 87 (3), 1651-4. Luo, S.; Kulak, J. M.; Cartier, G. E.; Jacobsen, R. B.; Yoshikami, D.; Olivera, B. M.; McIntosh, J. M., alpha-conotoxin AuIB selectively blocks alpha3 beta4 nicotinic acetylcholine receptors and nicotine-evoked norepinephrine release. The Journal of neuroscience: the official journal of the Society for Neuroscience 1998, 18 (21), 8571-9. Kulak, J. M.; McIntosh, J. M.; Yoshikami, D.; Olivera, B. M., Nicotine-evoked transmitter release from synaptosomes: functional association of specific presynaptic acetylcholine receptors and voltage-gated calcium channels. Journal of neurochemistry 2001, 77 (6), 1581-9.).
The α-CTxs having extraordinary selectivity to specific subtypes of nAChRs are necessary tools for studying distribution and functions of various subtypes and medicaments for treatment of associated diseases (Kasheverov, I. E., Utkin, Y. N., and Tsetlin, V. I. (2009) Naturally Occurring and Synthetic Peptides Acting on Nicotinic Acetylcholine Receptors. Current Pharmaceutical Design 15, 2430-2452; Nicke, A., Wonnacott, S., and Lewis, R. J. (2004) alpha-Conotoxins as tools for the elucidation of structure and function of neuronal nicotinic acetylcholine receptor subtypes. Eur. J. Biochem. 271, 2305-2319). The α-CTx being capable of specifically blocking α3β2 subtype, while having very small or even no activity of blocking very similar α6β2* subtype is very valuable, that is a ligand capable of distinguishing α3β2 and α6β2* subtypes has very important scientific and application value. The reason is that α6β2* subtype dominates dopaminergic region. Our knowledge about composition, properties and physiological functions of receptors in this important physiological region are merely from application of α-CTx MII, while α-CTx MII has poor selectivity to α3β2 and α6β2* subtypes, and cannot distinguishes them; or they are studied using selective blocking agents for α6β2* subtypes (Dowell, C., Olivera, B. M., Garrett, J. E., Staheli, S. T., Watkins, M., Kuryatov, A., Yoshikami, D., Lindstrom, J. M., and McIntosh, J. M. (2003) α-Conotoxin PIA Is Selective for 6 Subunit-Containing Nicotinic Acetylcholine Receptors. The Journal of Neuroscience 23, 8445-8452; McIntosh, J. M., Azam, L., Staheli, S., Dowell, C., Lindstrom, J. M., Kuryatov, A., Garrett, J. E., Marks, M. J., and Whiteaker, P. (2004) Analogs of alpha-conotoxin MII are selective for alpha 6-containing nicotinic acetylcholine receptors. Molecular pharmacology 65, 944-952; Quik, M., Perez, X. A., and Grady, S. R. (2011) Role of alpha 6 nicotinic receptors in CNS dopaminergic function: relevance to addiction and neurological disorders. Biochemical pharmacology 82, 873-882; Letchworth, S. R., and Whiteaker, P. (2011) Progress and challenges in the study of alpha 6-containing nicotinic acetylcholine receptors. Biochemical pharmacology 82, 862-872; Champtiaux, N., Gotti, C., Cordero-Erausquin, M., David, D. J., Przybylski, C., Lena, C., Clementi, F., Moretti, M., Rossi, F. M., Le Novere, N., McIntosh, J. M., Gardier, A. M., and Changeux, J. P. (2003) Subunit composition of functional nicotinic receptors in dopaminergic neurons investigated with knock-out mice. Journal of Neuroscience 23, 7820-7829). However, the expression of α3β2* nAChRs usually results in expression interlapping of α6β2* nAChRs, and α3β2* nAChRs are sometimes dominant (Whiteaker, P., McIntosh, J. M., Luo, S. Q., Collins, A. C., and Marks, M. J. (2000) I-125-alpha-conotoxin MII identifies a novel nicotinic acetylcholine receptor population in mouse brain. Molecular pharmacology 57, 913-925; Whiteaker, P., Peterson, C. G., Xu, W., McIntosh, J. M., Paylor, R., Beaudet, A. L., Collins, A. C., and Marks, M. J. (2002) Involvement of the alpha 3 subunit in central nicotinic binding populations. Journal of Neuroscience 22, 2522-2529; McClure-Begley, T. D., Wageman, C. R., Grady, S. R., Marks, M. J., McIntosh, J. M., Collins, A. C., and Whiteaker, P. (2012) A novel alpha-conotoxin MII-sensitive nicotinic acetylcholine receptor modulates H-3-GABA release in the superficial layers of the mouse superior colliculus. J Neurochem 122, 48-57). In addition, α3β2* nAChRs in spine also play an important role in transmission of pain stimulation and are analgesic action targets (Young, T., Wittenauer, S., McIntosh, J. M., and Vincler, M. (2008) Spinal α3β2* nicotinic acetylcholine receptors tonically inhibit the transmission of nociceptive mechanical stimuli. Brain research 1229, 118-124). Thus, finding a real α3β2* vs. α6β2* nAChRs selective blocking agent has very important value to comprehensively studying and understanding the functions and meanings of the subtype under normal and disease states.
It can be seen that α-conotoxins have tremendous potency for developing new medicines for treatment of pains, smoking cessation, rehabilitation, treatment of Parkinson's disease, dementia, depression and schizophrenia, and for studying mechanisms of associated diseases, and molecular probe tool drugs for distinguishing specific nAChRs subtypes as well as inventive drugs for treatment of neuralgia and addiction are also in urgent need to relieve damage and serious social problems caused by pains, smoking addiction and drug addiction. At present, it is still in urgent need to develop new nAChRs blocking agents with high specificity.
Contents of the Invention
After intensive study and creative efforts, the inventors of the present invention find a new type of α-conotoxin peptides, which can specifically block acetylcholine receptor, especially have strong activity of blocking a neuralgic drug target α3β2 nAChRs, α3β4 nAChRs or α6/α3β4 nAChRs, and an addictive drug target α6/α3β2β3 nAChRs or α3β4 nAChRs, and show very potent analgesic activity in animal models, so have good application prospect in aspects of manufacturing a medicament for analgesia, smoking cessation and rehabilitation, preventing and treating depression, dementia, schizophrenia, Parkinson's disease, or using as neuroscientific tool drugs. Thus, the following invention is provided:
One aspect of the present invention relates to a peptide, which has an amino acid sequence as shown in Formula I:GCCSX1PX2CX3X4X5X6PX7X8CX9 (SEQ ID NO: 68)  Formula I
wherein,
X1 represents D or H,
X2 represents P, A or V,
X3 represents R, N or S,
X4 represents N, V or A,
X5 represents K, D, M or A,
X6 represents H or S,
X7 represents D, E or X7 is absent,
X8 represents L or I,
X9 represents G or X9 is absent;
optionally, the C-terminal of the polypeptide of Formula I is amidated.
The above amino acids D, H, P, A, V, R, N, S, K, M, H, E, L, I, G are abbreviations of amino acids, which have the meanings well known by those skilled in the art.
The amidation of C-terminal of the polypeptide of Formula I can also be represented by #, i.e., GCCSX1PX2CX3X4X5X6PX7X8CX9#. (SEQ ID NO: 68)
The present invention further relates to a polypeptide, which is or comprises the amino acid sequence of any one of the following items (1) to (3):
(1) an amino acid sequence as shown in any one of sequences of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 11-15, SEQ ID NO: 26-28 or SEQ ID NO: 30;
(2) an amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, especially preferably at least 95%, most preferably at least 97% identity with the amino acid sequence of (1); or
(3) an amino acid sequence different from the sequence of (1) or (2) in substitution, deletion, insertion and/or addition of 1-5, preferably 1-3, more preferably 1-2, most preferably 1 amino acid residue.
For one purpose of the present invention, identity of two or more amino acid sequences is determined by BLAST2.0 Protein Database Query Program (Aaltschul et al., 1997, Nucleic Acid Research 25: 3389-3402) using the following parameters: blastall -p blastp-a4-e10-E0-v500-b250-I [query document]-d prot_all, in which -p refers to the name of program, -a refers to number of servers, -e refers to expectancy value, -E refers to cost of extension gap, -v refers to number of one-line description, -b refers to comparison number to be displayed, -I refers to query document, -d refers to database used for query.
The differences between the amino acid sequence of homologic polypeptide and amino acid sequence of any one of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 11-15, SEQ ID NO: 26-28 or SEQ ID NO: 30 may lie in substitution, insertion addition and/or deletion of 1 or more, preferably 1-5, more preferably 1-3, especially preferably 1-2, most preferably 1 amino acid residue. Preferably, the change of amino acid is a change having little effect on the property, i.e., it is a conservative amino acid substitution, a deletion of small fragment which usually is a deletion of 1 to about 5, preferably 1-3, more preferably 1 amino acid, a small amino or carboxyl terminal extension such as a methionine residue added to amino terminal, a small linker peptide having up to about 20-25 residues; or a small extension contributing to purification via changing net charge or other function such as polyhistidine fragment, epitope, binding domain, all of which do not significantly affect folding and/or activity of protein.
An example of conservative substitution is a substitution within basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophane and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). The amino acid substitutions usually not changing specific activity are known in the art, and are described in, for example, “Proteins”, H. Neurath and R. L. Hill, 1979, Academic Press, New York. The commonest substitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly, etc., and reversely performed substitutions.
The present invention further comprises fused polypeptides or lysable fused polypeptides in which the N-terminal and/or C-terminal of α-conotoxin is fused with other peptide/polypeptide. The technology for generating fused polypeptides is known in the art, comprising linking a coding sequence coding the peptide of the present invention with a coding sequence coding the other peptide/polypeptide so that they are in one reading frame and the expression of the fused polypeptide is controlled by the same promoter and terminator.
The polypeptide according to any one of items of the present invention preferably has an amino acid sequence as shown in SEQ ID NO: 4 (α-LvIA/LvD21), or SEQ ID NO: 3 (this peptide is actually a propeptide of α-LvIA/LvD21).
In the polypeptide according to any one of items of the present invention, the C-terminal of the polypeptide is preferably amidated. The amidation can be carried out via artificial chemical synthesis, or via an amidation enzyme in vivo or in vitro.
In the polypeptide according to any one of items of the present invention, preferably, the 1st cysteine and the 3rd cysteine of the N-terminal of the polypeptide form a disulfide bond, and the 2nd cysteine and the 4th cysteine form a disulfide bond; or the 1st cysteine and the 4th cysteine of the N-terminal of the polypeptide form a disulfide bond, and the 2nd cysteine and the 3rd cysteine form a disulfide bond; or the 1st cysteine and the 2nd cysteine of the N-terminal of the polypeptide form a disulfide bond, and the 3rd cysteine and the 4th cysteine form a disulfide bond.
The polypeptide of the present invention is conotoxin; specifically, α-conotoxin.
The conotoxin can be extracted from Conus lividus or Conus textile produced in Hainan Province of China; or can be an amino acid of chemical synthesis (e.g., the methods of Examples 2-(1) to 2-(3)); or a polypeptide obtained by expressing its nucleotide via genetic recombination (the nucleotide sequence can be prepared by the methods of Examples 1-(1) to 1-(3) or by the methods for direct polypeptide artificial synthesis of Examples 2-(1) to 2-(3)); or by referring to the following method:
Another aspect of the present invention relates to a method for preparing the polypeptides of any one of items of the present invention, comprising the following steps:
1) synthesizing a linear polypeptide by ABI Prism 433a polypeptide synthesizer or by manual method, in which side-chain protecting groups of Fmoc amino acid are: Pmc (Arg), Trt or Acm (Cys), But (Thr, Ser, Tyr), OBut (Asp) and Boc (Lys);
2) cutting the linear polypeptide synthesized in step 1) from resin;
3) using glacial diethyl ether to precipitate and wash the linear polypeptide obtained in step 2), and recovering a crude product of linear polypeptide;
4) using a preparative reversed phase HPLC C18 column (Vydac) to purify the crude product of linear polypeptide obtained in step 3);
5) subjecting the product obtained in step 4) to two- or one-step oxidative folding.
Another aspect of the present invention relates to a polynucleotide which codes an amino acid sequence of the polypeptide of any one of items of the present invention.
Preferably, the polynucleotide of any one of items of the present invention is or comprises a nucleotide sequence selected from any one of the following items (1) to (3):
(1) a nucleotide sequence as shown in any one of sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 16-21, SEQ ID NO: 22-25, SEQ ID NO: 29 or SEQ ID NO: 31;
(2) a complementary sequence of the nucleotide sequence of (1);
(3) a nucleotide sequence capable of hybridizing with the nucleotide sequence of (1) under a stringent condition.
As for hybridization between polynucleotides, reference can be made to many documents in the art, including, for example, Molecular Cloning: A Laboratory Manual, Edition 2, Sambrook, etc., Cold Spring Harbor Laboratory Press, Cold Spring, 1989. The hybridization can use stringent conditions of various degrees, for example, moderately stringent conditions, moderately-highly stringent conditions, or highly stringent conditions. The more stringent the conditions are, the higher complementary degree required for forming double helix. The stringent degree can be controlled via temperature, probe concentration, probe length, ion strength, time, etc. For double-stranded DNA, the hybridization is performed at a temperature 20-25° C. lower than the melting temperature [Tm] of DNA heterozygote in 6×SSPE, 5×Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA overnight. The washing is usually performed as follows: at Tm-20° C. in 0.2×SSPE, 0.1% SDS, once, 15 minutes (washing under moderately stringent condition).
Another aspect of the present invention relates to a nucleic acid construct, which comprises the polynucleotide of any one of items of the present invention.
Another aspect of the present invention relates to an expression vector, which comprises the nucleic acid construct of the present invention.
Another aspect of the present invention relates to a transformed cell, which comprises the expression vector of the present invention.
Another aspect of the present invention relates to a fused protein, which comprises the polypeptide of any one of items of the present invention.
Another aspect of the present invention relates to a pharmaceutical composition, which comprises the polypeptide of any one of items of the present invention, or the fused protein of the present invention; optionally, which further comprises a pharmaceutically acceptable carrier or excipient.
Another aspect of the present invention relates to a method for blocking acetylcholine receptor, comprising the step of using an effective amount of the polypeptide or fused protein of any one of items of the present invention; specifically, said acetylcholine receptor is α3β2 acetylcholine receptor, α6/α3 β2β3 acetylcholine receptor or α3β4 acetylcholine receptor.
Another aspect of the present invention relates to a method for screening an inhibitor of an acetylcholine receptor or determining the subtype of an acetylcholine receptor, the method comprising: the step of contacting an acetylcholine receptor with the polypeptide or fused protein of any one of items of the present invention in the presence or absence of a candidate compound; specifically, said acetylcholine receptor is α3β2 acetylcholine receptor, α6/α3β2β3 acetylcholine receptor or α3β4 acetylcholine receptor. When the polypeptide or fused protein can specifically block α3β2 acetylcholine receptor (e.g., α-conotoxin LvIA/LvD21), can specifically block α6/α3β2β3 acetylcholine receptor (e.g., α-conotoxin TxIB/Txd4) r, or can specifically block α3β4 acetylcholine receptor (e.g., α-conotoxin TxIC/Txd1), it can be determined that the acetylcholine receptor is α3β2 subtype, α6β2* subtype (α6/α3β2β3 acetylcholine receptor) or α3β4 subtype acetylcholine receptor.
Another aspect of the present invention relates to a use of the polypeptide or fused protein of any one of items of the present invention in blocking acetylcholine receptor; specifically, the acetylcholine receptor is α3β2 acetylcholine receptor, α6/α3β2β3 acetylcholine receptor or α3β4 acetylcholine receptor.
Another aspect of the present invention relates to a use of the polypeptide or fused protein of any one of items of the present invention in the manufacture of a medicament or reagent for blocking acetylcholine receptor; specifically, said acetylcholine receptor is α3β2 acetylcholine receptor, α6/α3β2β3 acetylcholine receptor or α3β4 acetylcholine receptor.
Another aspect of the present invention relates to a use of the polypeptide or fused protein of any one of items of the present invention in the manufacture of a medicament for treatment and/or prophylaxis and/or adjuvant therapy of a nervous system disease, such as addiction, neuralgia, Parkinson's disease, or dementia, or a use in the manufacture of a drug for killing a pest, analgesia, smoking cessation, or addiction treatment; specifically, said neuralgia is induced by the following causes: cancers and chemotherapy of cancers, alcoholism, ischioneuralgia, diabetes mellitus, prosopalgia, sclerosis, herpes zoster, mechanical injury and surgical injury, AIDS, head nerve paralysis, drug poisoning, industrial pollution poisoning, lymphatic neuralgia, myeloma, multipoint motor neuralgia, chronic congenital esthesioneurosis, acute spontaneous neuralgia, squeezing neuralgia, angiitis, vasculitis, ischemia, uremia, children biliary liver disease, chronic respiratory disorder, complex neuralgia, multiple organ failure, sepsis/pyaemia, hepatitis, porphyria, avitaminosis, chronic liver diseases, primary biliary cirrhosis, hyperlipidemia, leprosy, Lyme arthritis, sensory perineuritis, allergies, etc.
Another aspect of the present invention relates to a method for treatment and/or prophylaxis and/or adjuvant therapy of nervous system diseases, such as pains, addiction of tobacco, alcohol and drugs, dementia, schizophrenia, central nerve disorder, epilepsy, Parkinson's disease, mental disorder, neuromuscular blockade, myasthenia gravis, depression, hypertension, arrhythmia, asthma, muscular flaccidity, apoplexy, breast cancer and lung cancer, or a method for killing a pest, analgesia, smoking cessation, or addiction treatment, comprising the step of administering an effective amount of the polypeptide (conotoxin peptide or propeptide thereof) or fused protein of the present invention or the pharmaceutical composition of the present invention; specifically, said addiction is induced by addictive substance such as nicotine, morphine, cocaine, alcohol; the neuralgia is induced by the following reasons: cancers and chemotherapy of cancers, alcoholism, ischioneuralgia, diabetes mellitus, prosopalgia, sclerosis, herpes zoster, mechanical injury and surgical injury, AIDS, head nerve paralysis, drug poisoning, industrial pollution poisoning, lymphatic neuralgia, myeloma, multipoint motor neuralgia, chronic congenital esthesioneurosis, acute spontaneous neuralgia, squeezing neuralgia, angiitis, vasculitis, ischemia, uremia, children biliary liver disease, chronic respiratory disorder, complex neuralgia, multiple organ failure, sepsis/pyaemia, hepatitis, porphyria, avitaminosis, chronic liver diseases, primary biliary cirrhosis, hyperlipidemia, leprosy, Lyme arthritis, sensory perineuritis, allergies, etc.
The conotoxin peptide of the present invention can exert effects by binding α3β2 acetylcholine receptor (nAChR), α6/α3β2β3 acetylcholine receptor or α3β4 acetylcholine, have analgesia effect, can be used for studying, diagnosis and treatment nervous system diseases such as addiction, neuralgia, Parkinson's disease, dementia, schizophrenia, depression, and as a useful molecular probe in studying. Affinity of different α-CTx to vertebrate receptor is diverse, for example, in several orders of magnitude. Such diversity among germ lines makes α-CTx be useful as a probe for studying phylogenesis of vertebrates, or be useful as a molecular probe for determining different subtypes of nAchRs. They are candidate drugs, primary drugs and therapeutic drugs in developing new drugs.
The terms used in the present invention are explained as follows.
Neuralgia
The polypeptide of the present invention relates to a use for treatment of various neuralgias. Neuralgia is a pain caused by a primary or secondary lesion or a functional disorder or a transient disorder of peripheral or central nervous system, manifesting in spontaneous pain, sense hypersensitivity, etc. Neuralgia may be caused by many diseases, including cancers and chemotherapy of cancers, alcoholism, ischioneuralgia, diabetes mellitus, prosopalgia, sclerosis, herpes zoster, mechanical injury and surgical injury, AIDS, head nerve paralysis, drug poisoning, industrial pollution poisoning, myeloma, multipoint neuralgia, chronic congenital esthesioneurosis, acute fierce spontaneous neuralgia, squeezing neuralgia, angiitis (vasculitis)/ischemia, uremia, children biliary liver disease, chronic respiratory disorder, complex neuralgia, multiple organ failure, sepsis/pyaemia, hepatitis, porphyria, avitaminosis, chronic liver diseases, primary biliary cirrhosis, hyperlipidemia, leprosy, Lyme arthritis, sensory perineuritis, allergies, etc.
Addiction
The polypeptide of the present invention relates to treatment of addiction caused by various dependent substances. Addiction refers to a periodical or chronic poisoning state of a subject who repeatedly uses a psychoactive substance. The psychoactive substance refers to nicotine, opium, heroine, methylamphetamine (ice), morphine, marihuana, cocaine, and other narcotics and psychotropic substances that can cause addiction in human and are controlled by national regulations. Addiction relates to the generation of a great amount of dopamine, exhibiting uncontainable use of a favorite substance or a use behavior that can hardly be restrained or rectified, and unscrupulously using any means to obtain psychoactive substances for acquiring good feel or avoiding withdrawal symptoms. Typical situations are increase of resistance and occurrence of withdrawal symptoms. The life of addict is totally mastered by the addictive substance and thus is seriously affected, and even rejects other important action and all responsibility. Hence, the use of addictive substance would bring bout damage to both individual and society. When using with alcohol, addiction is equivalent to chronic alcoholism. The term “addiction” also covers both physical and psychological contents. Psychological addiction emphasizes control impaired experience in alcohol drinking and drug administration, while physical addiction refers to resistance and withdrawal symptoms.
Nucleic Acid Construct
The present invention further relates to a nucleic acid construct comprising the nucleic acid sequence of the present invention and 1 or more regulatory sequences operably linked thereto, in which the regulatory sequences under compatible conditions thereof can guide an encoding sequence to express in a suitable host cell. The expression should be understood to comprise any steps relating to produce polypeptide, including but not limited to transcription, modification after transcription, translation, modification and secretion after translation.
In the text, “nucleic acid construct” is defined as a single chain or double chain nucleic acid molecule, which is separated from natural gene, or comprises nucleic acid fragments combined and collocated in non-natural manner via modification. When the nucleic acid construct comprises all regulatory sequences necessary for expressing the coding sequence of the present invention, the term “nucleic acid construct” has the same meaning of expression kit. In the text, the term “coding sequence” is defined as a part of nucleic acid sequence for directly determining amino acid sequence of protein product. The boundaries of coding sequence usually are determined with ribosome bind site (corresponding to prokaryotic cell) closely adjacent to mRNA 5′ terminal open reading frame upstream and transcription termination sequence closely adjacent to mRNA 3′ terminal open reading frame downstream. Coding sequence can comprise but is not limited to DNA, cDNA and recombinant nucleic acid sequence.
The separated nucleic acid sequence encoding the peptide of the present invention can be manipulated in many manners so as to express the peptide. Depending on expression vector, the nucleic acid sequence can be processed before insertion into the vector if necessary. The technology of modifying nucleic acid sequence using recombinant DNA method is well known in the art.
The term “regulatory sequences” in the text is defined as all components necessary for or contributive to the expression of the peptide of the present invention. Each regulatory sequence naturally exists in or is extraneously added to the nucleic acid sequence coding the polypeptide. These regulatory sequences include but are not limited to leader sequences, polyadenylate sequences, propeptide sequences, promoters, signal sequences and transcription terminators. The lowest limit is that the regulatory sequences should comprise promoters and termination signals for transcription and translation. In order to introduce a specific restriction site to link the regulatory sequence to the coding region of nucleic acid sequence coding the polypeptide, a regulatory sequence with a connector can be provided. The term “operably linking” in the text refers to a conformation in which the regulatory sequence is at a suitable position of coding sequence corresponding to DNA sequence so that the regulatory sequence guides the expression of the polypeptide.
The regulatory sequence can be any suitable promoter sequence, i.e., a nucleic acid sequence that can be recognized by a host cell expressing nucleic acid sequence. The promoter sequence comprises a transcriptional regulatory sequence mediating polypeptide expression. The promoter can be any nucleic acid sequence having transcription activity in a selected host cell, including mutant, truncated and hybridized promoters, and can be obtained from a gene coding extracellular or intracellular polypeptide homologous or heterogeneous to host cell.
The regulatory sequence can further be a suitable transcription termination sequence, i.e., a sequence capable of being recognized by a host cell so as to terminate transcription. The termination sequence is operably linked to 3′ terminal of the nucleic acid sequence coding the polypeptide. Any terminators having such function in a selected host cell can be used in the present invention.
The regulatory sequence can further be a suitable leader sequence, i.e., a mRNA untranslated region very important for translation of host cell. The leader sequence is operably linked to 5′ terminal of the nucleic acid sequence coding the polypeptide. Any leader sequence capable of exerting the function in a selected host cell can be used in the present invention.
The regulatory sequence can further be a coding region of signal peptide, and the region codes an amino acid sequence linked to amino terminal of polypeptide, and can lead the coded polypeptide into cell secretion route. The 5′ terminal of coding region of nucleic acid sequence can naturally contain a signal peptide coding region consistent to translation reading frame and naturally linked to a coding region of secreted polypeptide. Or, the 5′ terminal of coding region can contain extraneous signal peptide coding region relative to the coding sequence. When the coding sequence does not contain signal peptide coding region under normal condition, an extraneous signal peptide coding region may be added. Or, an extraneous signal peptide coding region can be used to simply substitute a natural signal peptide coding region so as to enhance secretion of polypeptide. However, any signal peptide coding region capable of leading an expressed polypeptide to enter into a secretion route of a used host cell can be used in the present invention.
The regulatory sequence can further be a propeptide coding region, and the region codes an amino acid sequence at amino terminal of polypeptide. The obtained polypeptide is called as proenzyme or propolypeptide. The propolypeptide usually has not activity, and can be transformed into a mature active polypeptide by cutting propeptide from propolypeptide via catalysis or self-catalysis.
When the amino terminal of polypeptide has both signal peptide and propeptide, the propeptide is close to the amino terminal of polypeptide, while the signal peptide is close to the amino terminal of the propeptide.
It may also be necessary to add a regulatory sequence capable of regulating polypeptide expression according to growth conditions of host cells. Examples of regulatory system are systems capable of responding to a chemical or physical stimulation (included in a condition having a regulatory compound) so as to open or close gene expression. Other examples of the regulatory sequence are regulatory sequences capable of amplifying gene. In these examples, the nucleic acid sequence coding polypeptide should be operably linked to the regulatory sequence.
Expression Vector
The present invention further relates to a recombinant expression vector comprising the nucleic acid sequence of the present invention, a promoter and a terminal signal for transcription and translation. The above nucleic acids and regulatory sequences could be linked together to prepare a recombinant expression vector, and the vector can comprise 1 or more convenient restriction sites so that the nucleic acid sequence coding the polypeptide can be inserted or substituted at these sites. Or, the nucleic acid sequence or a nucleic acid construct comprising the sequence can be inserted into a suitable expression vector to express the nucleic acid sequence of the present invention. When the expression vector is prepared, the coding sequence can be in the vector so as to operably link to a suitable expression regulatory sequence.
The recombinant expression vector can be any vector (e.g., plasmid or virus) capable of performing recombinant DNA operation and expressing nucleic acid. The selection of vector usually depends on compatibility of vector and host cell into which the vector is introduced. The vector can be a linear or closed plasmid.
The vector can be an autonomously replicating vector (i.e., an extrachromosomal complete construct, which can be replicated independent of chromosome), such as plasmid, extrachromosomal component, minute chromosome, or artificial chromosome. The vector can comprise any mechanism ensuring self-replication. Or, the vector is a vector that is integrated into genome and replicated together with the chromosome into which it is integrated when the vector is introduced into a host cell. In addition, the used vector can be a single vector or plasmid, or generally contained 2 or more vectors or plasmids of total DNA to be introduced into host cell genome, or a transposon.
Preferably, the vector of the present invention comprises 1 or more selective markers convenient for selecting transformed cells. The selective marker is such a gene which product provides a resistance against a biocide, a resistance against a heavy metal, or provides an auxotroph prototrophy. Examples of bacterial selective markers are daI gene of bacillus subtilis or bacillus licheniformis, or resistance makers including antibiotics such as ampicillin, kanamycin, chloromycetin, or tetracycline.
Preferably, the vector of the present invention comprises components ensuring the vector to be stably integrated into genome of host cell, or ensuring the vector to be autonomously replicated independent to cell genome in cell.
As to autonomous replication, the vector can further comprise a replication organ so that the vector can be autonomously replicated in host cell. The replication organ can have a mutation that makes it a temperature-sensitive type in the host cell (see: for example, fEhrlich, 1978, National Academy of Sciences, 75:1433).
More than one copy of the nucleic acid sequence of the present invention can be inserted into a host cell to increase the output of gene product. The number of copies of the nucleic acid sequence can be increased by inserting at least one additional copy of the sequence into genome of host cell, or by inserting the nucleic acid sequence together with an amplification selective marker, culturing cells in the presence of a suitable selective reagent, picking out cells that have selective marker gene for copy amplification and thus have additional copies of the nucleic acid.
The steps for linking the above components to construct the recombinant expression vector of the present invention are well known in the art (see: for example, Molecular Cloning: A Laboratory Manual, Edition 2, Sambrook, etc., Cold Spring Harbor Laboratory Press, Cold Spring, 1989).
Host Cells
The present invention further relates to a recombinant host cell comprising the nucleic acid sequence of the present invention for recombination production of polypeptide. A vector comprising the nucleic acid sequence of the present invention can be introduced into a host cell so that the vector is maintained in form of the above chromosomal integrated body or self-replicable extrachromosomal vector. The term “host cell” covers any offspring that are different from parent cells due to mutation during replication period. The selection of host cell mainly depends on polypeptide coding gene and source thereof.
The host cell can be a prokaryotic cell or an eukaryotic cell, for example, a bacterium or yeast cell. The vector can be introduced into the host cell by a technology well known in the art.
Preparation Method
The present invention further relates to a method for recombination production of the peptide of the present invention, the method comprising: (a) culturing a host cell having a nucleic acid construct under conditions suitable to produce the peptide, the nucleic acid construct comprising a nucleic acid sequence encoding the peptide; and (b) recovering the peptide.
In the preparation method of the present invention, the cell is cultured in a nutrient medium suitable for polypeptide production by a method known in the art. For example, the cell is cultured by shake-flask culture, laboratory culture, small or large scale fermentation in industrial fermentation tank (including continuous, batch, batch charging or solid state fermentation) in a suitable culture medium under conditions allowing polypeptide expression and/or separation. The culture can be carried out with steps known in the art in a suitable culture medium containing carbon source and nitrogen source and inorganic salt. The suitable culture medium can be provided by suppliers or prepared according to a composition known in the art (for example, those in the catalogue of American Type Culture Collection). If the polypeptide is secreted in the culture medium, the polypeptide can be directly recovered from the culture medium. If the polypeptide is not secreted, it can be recovered from cell lysate.
The produced polypeptide can be recovered by a method known in the art. For example, the polypeptide can be recovered from the culture medium by conventional steps (including but not limited to centrifugation, filtration, spray drying, evaporation or precipitation).
The polypeptide of the present invention can be purified by known steps in the art, and these steps include but are not limited to chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatofocusing, and size exclusion chromatography), HPLC, electrophoresis (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE or extraction (see: for example, Protein Purification, edited by J. C. Janson and Lars Ryden, VCH Publishers, New York, 1989).
Transgenic Animals and Plants
The present invention further relates to an animal or plant cell transformed with the nucleic acid sequence of the present invention, preferably a plant cell of wheat, maize, so as to give the transformed host a new property (e.g., pest resistance). This can be fulfilled by transforming the animal or plant cell with the construct disclosed in the present invention by a method well known in the art.
Method and Preparation for Controlling Pests
Many methods known by those skilled in the art can be used for controlling pests with the conotoxin peptide or polynucleotide of the present invention. These methods comprise, for example, applying a recombinant microorganism to pests (or their locus), and transforming a plant with a gene encoding the conotoxin peptide of the present invention. The transformation can be carried out by conventional methods known by those skilled in the art. Necessary substances for such transformation are disclosed here or can be readily obtained via other routes by those skilled in the art.
The preparation containing the conotoxin peptide or the recombinant microorganism of the polynucleotide of the present invention can be applied to soil. The prepared product can further be used for seed coating or root treatment or application on whole plant in later period of plant growth cycle. The preparation can comprise a diffusion-thickening adjuvant, a stabilizing agent, other pesticide additives, or a surfactant. A liquid preparation can be aqueous or nonaqueous, and used in form of foam, gel, suspension, emulsible concentrate. Components can comprise rheological agents, surfactants, emulsifying agents, dispersing agents, or polymers.
Those skilled in the art understand that pesticide can have a widely variable concentration due to nature of specific preparations, especially, it can be a concentrate or directly used. Pesticide can be in an amount of at least 1% by weight, or 100% by weight. Dry preparation usually has about 1-95% by weight of pesticide, while liquid preparation usually has a solid content of about 1-60% by weight in liquid phase. A preparation containing cells usually have about 102 to about 104 cells/mg. These preparations can be applied in an amount of 50 mg (liquid or dry) to 1 kg per hectare. The preparations can be applied to pest environment such as soil and plant by spraying, scattering, splashing.
Pharmaceutical Composition
The present invention further relates to a pharmaceutical composition comprising the peptide of the present invention and a pharmaceutically acceptable carrier and/or excipient. The pharmaceutical composition can be used for studying, diagnosis, alleviation or treatment of diseases or disorders relating to addiction, neuralgia, mental retardation, pain, Parkinson's disease, mental disorders, depression, myasthenia gravis, cancers, etc. In an embodiment, a pharmaceutical composition comprising a therapeutically effective amount of the peptide of the present invention is prepared and administered in a way facilitating medicinal application, while clinical state of individual patient, delivery site, administration method, administration schedule and other factors known by doctor should also be considered. Thus, “effective amount” for the purpose in the text is determined with consideration in these aspects.
A pharmaceutical composition comprising a therapeutically effective amount of the peptide of the present invention can be administered parenterally, orally, intracisternally, intrathecally. “Pharmaceutically acceptable carrier” refers to a nontoxic solid, semi-solid or liquid filler, diluent, capsule material or any type of formula assistants. The term “parenterally” in the text refers to administration manners including intravenous, intramuscular, intraperitoneal, intrathoracic, subcutaneous, intrathecal, and intra-articular injection or infusion. The polypeptide of the present invention can also be administered via a sustained-release system.
The present invention further relates to a pharmaceutical composition for specifically blocking nAChRs.
The conotoxin peptide of the present invention can be used as a probe for studying phylogenesis of animal nAChRs; as a probe for determining different subtypes of nAChRs; as a molecular model for designing new drug; as a tool drug and treatment drug for studying and diagnosis of neurological diseases such as addiction, Parkinson's diseases, dyspraxia, schizophrenia; a candidate drug for treatment of breast cancer, lung cancer, small cell lung cancer, or as a polypeptide pesticide for developing a new type of biopesticide.
Beneficial Effects of the Invention
The α-conotoxin peptide of the present invention can specifically block acetylcholine receptors (nAChRs), and has potent activity of analgesia and addiction withdrawal, and functions for treatment of Parkinson's disease, breast cancer and lung cancer cells, as well as functions for treatment of diseases such as addiction, dementia, schizophrenia, depression.